Published on Web 12/10/2004
Chain-Length-Dependent Helical Motifs and Self-Association of β-Peptides with Constrained Side Chains Anaszta´ zia Hete´ nyi,† Istva´ n M. Ma´ ndity,† Tama´ s A. Martinek,† Ga´ bor K. To´ th,‡ and Ferenc Fu¨lo¨p*,† Contribution from the Institute of Pharmaceutical Chemistry and Department of Medical Chemistry, UniVersity of Szeged, P.O. Box 121, H-6701 Szeged, Hungary Received April 29, 2004; E-mail:
[email protected] Abstract: Homo-oligomers constructed by using trans-2-aminocyclohexanecarboxylic acid monomers without protecting groups were studied. Both ab initio theory and NMR measurements showed that the tetramer tends to adopt a 10-helix motif, while the pentamer and hexamer form the known 14-helix. It was concluded that the conformationally constrained backbone is flexible enough to afford both 10-helical and 14-helical motifs, this observation in turn providing evidence of the true folding process. Self-association of the helical units was also detected, and the results of variable-temperature diffusion NMR measurements strongly suggested the presence of helical bundles in methanol solution.
Introduction
The artificial secondary structures of β-peptides are of major importance in the field of self-organizing systems by virtue of the wide range of their potential applications, due to their propensity to adopt side-chain-controllable compact ordered conformations.1-6 The conformationally constrained β-peptide oligomers containing cyclic side chains are among the most thoroughly studied models in foldamer chemistry. Homooligomers constructed by using trans-2-aminocyclohexanecarboxylic acid (trans-ACHC) monomers, which have protecting groups at both ends, 1-5 (Chart 1), are known to form a highly stable 14-helix in various solvents (methanol or pyridine).7-15 This structural motif is so stable that it is always predominant when the chain is longer than three monomers. The chain-length † ‡
Institute of Pharmaceutical Chemistry. Department of Medical Chemistry.
(1) Hill, D. J.; Mio, M. J.; Prince, R. B.; Hughes, T. S.; Moore, J. S. Chem. ReV. 2001, 101, 3893-4011. (2) Martinek, T. A.; Fu¨lo¨p, F. Eur. J. Biochem. 2003, 270, 3657-3666. (3) Venkatraman, J.; Shankaramma, S. C.; Balaram, P. Chem. ReV. 2001, 101, 3131-3152. (4) Saven, J. G. Chem. ReV. 2001, 101, 3113-3130. (5) Seebach, D.; Abele, S.; Gademann, K.; Guchard, G.; Hintermann, T.; Jaun, B.; Matthews, J. L.; Schreiber, J. V. HelV. Chim. Acta 1998, 81, 932-982. (6) (a) Epand, R. F.; Raguse, T. L.; Gellman, S. H.; Epand, R. M. Biochemistry 2004, 43, 9527-9535. (b) Schmitt, M. A.; Weisblum, B.; Gellman, S. H. J. Am. Chem. Soc. 2004, 126, 6848-6849. (7) Appella, D. H.; Christianson, L. A.; Karle, I. L.; Powell, D. R.; Gellman, S. H. J. Am. Chem. Soc. 1996, 118, 13071-13072. (8) Appella, D. H.; Christianson, L. A.; Karle, I. L.; Powell, D. R.; Gellman, S. H. J. Am. Chem. Soc. 1999, 121, 6206-6212. (9) Appella, D. H.; Christianson, L. A.; Klein, D. A.; Richards, M. R.; Powell, D. R.; Gellman, S. H. J. Am. Chem. Soc. 1999, 121, 7574-7581. (10) Barchi, J. J., Jr.; Huang, X.; Appella, D. H.; Christianson, L. A.; Durrell, S. R.; Gellman, S. H. J. Am. Chem. Soc. 2000, 122, 2711-2718. (11) Appella, D. H.; Christianson, L. A.; Klein, D. A.; Powell, D. R.; Huang, X.; Barchi, J. J.; Gellman, S. H. Nature 1997, 387, 381-384. (12) Gellman, S. H. Acc. Chem. Res. 1998, 31, 173-180. (13) Appella, D. H.; Barchi, J. J.; Durell, S. R.; Gellman, S. H. J. Am. Chem. Soc. 1999, 121, 2309-2310. (14) Raguse, T. L.; Lai, J. R.; LePlae, P. R.; Gellman, S. H. Org. Lett. 2001, 3, 3963-3966. (15) Martinek, T. A.; To´th, G. K.; Vass, E.; Hollo´si, M.; Fu¨lo¨p, F. Angew. Chem., Int. Ed. 2002, 41, 1718-1721. 10.1021/ja0475095 CCC: $30.25 © 2005 American Chemical Society
Chart 1
independence of the folding pattern raises the still open question of whether these oligomers exhibit a real folding process, or whether the conformational space of the monomers is too preorganized to allow partly folded or other stable secondary structures. The torsional angles φ, θ, and ψ of the β-residues extracted from the literature data1,5,16-18 for the 10- and 14helices with the same helicity occupy adjacent positions in the Ramachandran plot, which allows a smooth rearrangement between the two conformational states without unfolding of the helical pattern if the 10-helix is energetically available under the corresponding conditions. Accordingly, by utilizing circular dichroism (CD) spectra together with molecular dynamics simulation, Seebach et al. proposed an equilibrium between the 10- and 14-helices in β-hexapeptide derivatives with simple proteinogenic side chains19 while Fleet et al. demonstrated experimentally that an oxetane-based β-residue leads directly to the 10-helix formation.20 Such a conformational polymorphism is an important feature of any folded system that is (16) Banerjee, A.; Balaram, P. Curr. Sci. India 1997, 73, 1067-1077. (17) Seebach, D.; Gademann, K.; Schreiber, J. V.; Matthews, J. L.; Hintermann, T.; Jaun, B.; Oberer, L.; Hommel, U.; Widmer, H. HelV. Chim. Acta 1997, 80, 2033-2038. (18) Gu¨nther, R.; Hofmann, H. J. HelV. Chim. Acta 2002, 85, 2149-2168. (19) (a) Seebach, D.; Schreiber, V. J.; Abele, S.; Daura, X.; Gunsteren, F. W. HelV. Chim. Acta 2000, 83, 34-57. (b) Daura, X.; Bakowies, D.; Seebach, D.; Fleischhauer, J.; van Gunsteren, W. F.; Kru¨ger, P. Eur. Biophys. J. 2003, 32, 661-670. (20) Claridge, D. W. T.; Goodman, M. J.; Moreno, A.; Angus, D.; Barker, F. S.; Taillefumier, C.; Watterson, P. M.; Fleet, W. J. G. Tetrahedron Lett. 2001, 42, 4251-4255. J. AM. CHEM. SOC. 2005, 127, 547-553
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designed to have a complex dynamic function in general; it is also observed for the natural R-peptides: the interplay between the R-helix and the 310-helix motif can be a crucial factor during the folding process.21 To reveal similar intrinsic properties of the β-peptides and experimentally capture the conformational polymorphism in high-resolution solution structures, short oligomers with conformationally constrained side chains without terminal protecting groups were studied in the present work. We set out to discover the limits of the flexibility of transACHC oligomers and to test the ability of these oligomers to adopt the 10-helix motif besides the well-known 14-helix, which would suggest that the 10-helix may be a potential conformational intermediate in the folding process toward the thermodynamically stable 14-helix. Results and Discussion
In our approach, the potential energy hypersurfaces of transACHC homo-oligomers without protecting groups, 6-9 (Chart 1), were probed first by a standard restrained simulated annealing conformational search protocol, carried out by using molecular mechanics and distance restraints between the relevant H-bond pillar atoms. The resulting most stable structures for 7-9 were optimized without restraints for both the 10- and 14helices. Final minimizations were carried out by using an ab initio quantum mechanical method22 at the RHF/3-21G level in a vacuum, as this has been reported to provide a good approximation for the geometry of the β-peptides.23 The ab initio structures converged to the corresponding local minimum of the potential energy surface. To take into account the effects of more diffuse basis sets and the electron correlation, the energies were calculated at the B3LYP/6-311G** level too (Figure 1). The conformational energy differences between the 10- and 14helices (∆E ) E10 - E14) proved to be rather low in terms of the nonbonding interaction energy scale, indicating a possible energetic availability of the 10-helix. The ∆E values clearly reveal a trend of an increasing relative stability for the 10-helix motif as the chain length decreases. The energy difference obtained with the different approximation levels for 7 predict a conformational equilibrium between the 10- and 14-helices, with a slight preference for the 10-helix in the case of the RHF/321G level. These findings presage the tendency for the unprotected tetramer to adopt the 10-helical motif, which is surprising in light of the earlier literature results on similar constrained β-peptides with protecting groups and offers a possibility for experimental testing. (21) Bolin, A. K.; Millhauser, L. G. Acc. Chem. Res. 1999, 32, 1027-1033. (22) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Montgomery, J. A., Jr.; Vreven, T.; Kudin, K. N.; Burant, J. C.; Millam, J. M.; Iyengar, S. S.; Tomasi, J.; Barone, V.; Mennucci, B.; Cossi, M.; Scalmani, G.; Rega, N.; Petersson, G. A.; Nakatsuji, H.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Klene, M.; Li, X.; Knox, J. E.; Hratchian, H. P.; Cross, J. B.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Ayala, P. Y.; Morokuma, K.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Zakrzewski, V. G.; Dapprich, S.; Daniels, A. D.; Strain, M. C.; Farkas, O.; Malick, D. K.; Rabuck, A. D.; Raghavachari, K.; Foresman, J. B.; Ortiz, J. V.; Cui, Q.; Baboul, A. G.; Clifford, S.; Cioslowski, J.; Stefanov, B. B.; Liu, G.; Liashenko, A.; Piskorz, P.; Komaromi, I.; Martin, R. L.; Fox, D. J.; Keith, T.; Al-Laham, M. A.; Peng, C. Y.; Nanayakkara, A.; Challacombe, M.; Gill, P. M. W.; Johnson, B.; Chen, W.; Wong, M. W.; Gonzalez, C.; Pople, J. A. Gaussian 03, Revision A.1; Gaussian, Inc.: Pittsburgh, PA, 2003; http://www.gaussian.com. (23) (a) Beke, T.; Csizmadia, I. G.; Perczel, A. J. Comput. Chem. 2004, 25, 285-307. (b) Mohle, K.; Gunther, R.; Thormann, M.; Sewald, N.; Hofmann, H. J. Biopolymers 1999, 50, 167-184. 548 J. AM. CHEM. SOC.
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Figure 1. Some of the resulting ab initio structures, and the chain-length dependence of the conformational energy difference between the 10- and 14-helices: [, RHF/3-21G; 2, B3LYP/6-311G**.
The Boc-(1S,2S)-ACHC monomer was synthesized by a literature method.24 The chain assembly was carried out in the solid phase; after the third amino acid, the acyl fluoride method25-27 was used, due to the difficulty of elongation of the peptide chain. The peptides were detached from the resin by using liquid HF, and the product was isolated by RP-HPLC. Products were characterized by means of various NMR methods, including COSY, TOCSY, and ROESY, in 8 mM CD3OD and DMSO solutions. For assessment of the conformational stability of the peptides in CD3OD, 1H-2D amide proton exchange was utilized. For 6, an unfolded conformation was found, as deciphered from the immediate amide proton exchange. Following the dissolution of 7, 1H NMR indicated that the amide protons exchanged completely only in 2 weeks. The amide protons in 8 and 9 still showed strong resonances after the same period of time. The shielded amide protons of the residues in the corresponding oligomers were assigned (Figure 2). The observations confirmed that the studied oligomers adopt very stable intramolecular H-bonded folding patterns in CD3OD solution. As concerns the previously reported trans-ACHC oligomers with protecting groups, the amide proton signals were lost within 65 h,7 which may suggest considerably more shielded NH protons in our models, but a direct comparison must be made with caution because of the incidentally different concentrations of residual acid (TFA) stemming from the chromatographic separation and sample preparation protocol. The 1H-2D amide proton exchange experiments were therefore repeated for 9 with an elevated TFA concentration of 1.0% v/v. The residual signals of the most shielded protons exhibit a relative intensity of 0.2 after 65 h for 9 (Figure 3). The usual TFA concentration during HPLC separations is 0.1% v/v; hence, the applied TFA level in these 1H-2D exchange experiments is drastically higher than one would expect in a normal setup. Accordingly, these results still support the view of highly shielded and stable H-bonds in the studied compounds. (24) Kanerva, L. T.; Csomo´s, P.; Sundholm, O.; Berna´th, G.; Fu¨lo¨p, F. Tetrahedron: Asymmetry 1996, 7, 1705-1716. (25) Carpino, L. A.; Elfaham, A. J. Am. Chem. Soc. 1995, 117, 5401-5402. (26) Wenschuh, H.; Beyermann, M.; Winter, R.; Bienert, M.; Ionescu, D.; Carpino, L. A. Tetrahedron Lett. 1996, 37, 5483-5486. (27) Carpino, L. A.; Beyermann, M.; Wenschuh, H.; Bienert, M. Acc. Chem. Res. 1996, 29, 268-274.
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Figure 3. Comparison of the NH/ND exchange rates: (a) without additional TFA; (b) with 1% TFA. The displayed intensities are assigned as follows: ×, NH2; O, NH3; +, NH4; 4, NH5; /, NH6. Figure 2. NH/HD exchange results for 7-9 in CD3OD.
The high-resolution 3D structure assignment was carried out by running ROESY spectra with 225 ms (Figure 4) and 400 ms spin lock in CD3OD. The intensity ratio (R) of the crosspeaks NHi-CRHi-1 and NHi-CβHi calculated according to eq 1, where INOE designates the integrated and offset-compensated
R ) INOE(NHi-CRHi-1)/INOE(NHi-CβHi)
(1)
ROESY cross-peak intensities, is a sensitive indicator of the overall fold of the oligomers because it is dependent on the sixth power of the relative distances.15 By using secondary structure motifs of (1S,2S)-ACHC obtained by molecular modeling, representative R values are estimated as 3.51, 5.83, and 5.61 for the right-handed 12-helix, left-handed 10-helix, and left-handed 14-helix, respectively. The random coil gives an R value of 2.1. It is clear that the 10- and 14-helices cannot be distinguished in this way, considering their similar R values and the usual experimental error; nevertheless, the overall fold can be determined. Comparison of the experimental intensity ratios and the theoretical values reveals good agreement with the left-handed 10- or 14-helical fold for each compound, with the exception of the N-terminal residues (Table 1). For 7, the NH2-CRH1 NOE interaction is missing (i.e., R ) 0, Figure S6 in the Supporting Information), which is a major deviation from the regular helix structure, pointing to a fraying or unusually oriented N-terminal residue. This observation is supported by the relatively faster exchange rate of NH2 and the considerable upfield shift of CRH1
(2.14 ppm) in comparison with those for 8 (2.96 ppm) and 9 (2.95 ppm). For 8 and 9, the R values involving the first residue are slightly decreased, but there is not a dramatic difference. The antiperiplanar arrangement of the NHi-CβHi protons in both the 10- and 14-helices was confirmed by the uniform 3J(NHiCβHi) values of ca. 9 Hz. As regards the theoretical models, another major difference between the two helical structures is that the 10-helix should give CRHi-CβHi+2 and the 14-helix CRHi-CβHi+3 NOE interactions. For 7, a marked signal can be observed between the protons at 2.85 ppm, unambiguously assigned to CRH2, and at 4.02 ppm. The resonances of CβH2 and CβH4 overlap near 4.02 ppm, but analysis of the vicinal coupling constant pattern clearly demonstrates the prevailing trans-diaxial orientation of CβH2 and CRH2, which rules out their proximity of
